Pentamycin, like Amphotericin B and Nystatin A1, belongs to the class of polyene macrolide antibiotics having antifungal activity. Pentamycin is obtainable from natural sources, e.g. it may be isolated from certain Streptomyces strains, like the mycelium of the actinomyces Streptomyces penticus as described by S. Umezawa and Y. Tanaka in J. Antibiotics, Ser. A, vol. XI, no. 1, pages 26 to 29 (1958), or from Streptomyces roseo luteus (NRRL 2776, NCIB 8984) as described in British patent 884711 to Glaxo. Said patent is directed to the production of the antibiotic lagosin which has been shown in the below-mentioned Pandey et al. article in J. Antibiotics vol. XXXV no. 8, pages 988-996 (1982) to be identical to fungichromin and cogomycin. Lagosin, both as a solid and in solution is stated in said British patent, page 2, right column, lines 48-49, to be sensitive to light.
The absolute configuration of pentamycin (=fungichromin) was determined by spectral comparison of the degradation products and partial synthesis by T. Oishi, Pure & Appl. Chem., vol. 61, no. 3, pp. 427-430 (1989). According to an X-ray analysis published by Y. Igarashi et al. in J. Antibiot. vol. 58, no. 8, pp. 523-525 (2005) pentamycin has the following formula

As stated e.g. on page 478, left column, of an article by David R. Worthen et al., Drug Development and Industrial Pharmacy, vol. 27(4), 277-286 (2001) “the production and purification of polyene antifungals are confounded by their inherent chemical instability. The polyenes typically possess one or several potentially unstable structural functionalities, including hydrolyzable esters, acetals, and hemiacetals, as well as conjugated polyene systems vulnerable to oxidation (19). Thus, all of the polyene antifungals, to a certain extent, are subject to inactivation or frank degradation by conditions routinely encountered during their production in and recovery from mass culture. Most notable of these potentially degradative conditions include moisture, elevated temperature, atmospheric oxygen, polyvalent metals, and exposure to light (19, 20).” The references 19 and 20 mentioned in said article are (19) I. M. Teresin, Polyene Antibiotics—Present and Future; University of Tokyo Press: Tokyo, 1976; 122-123, and (20) K. Thoma and N. Kubler, Photostability of antifungal agents. 2. Photostability of polyene antibiotics. Pharmazie vol. 52,294-302 (1997).
The above-mentioned Worthen article goes on by stating on page 478, left column, that “further complicating polyene purification is the fact that virtually all crude polyene isolates from Streptomyces contain several distinct, although physicochemically similar, isoforms, only one of which may be desirable for clinical use.”
In view of the above, it is not surprising that pentamycin was also given three other names, i.e. lagosin, fungichromin, and cogomycin because they were initially thought to be different substances. As stated by R. C. Pandey et al., J. Antibiotics vol. XXXV no. 8, pages 988-996 (1982) in the abstract “The three polyene macrolide antibiotics, fungichromin, lagosin, and cogomycin, previously described as having some stereochemical differences at one or more centers, are shown by . . . to be identical in all respects, including stereochemical aspects. The differences observed earlier in their properties have now been ascribed to varying amounts of impurities which are separable by high-performance liquid chromatography. All three antibiotics contain one major and several minor components” (emphasis added). In table I on page 988 of said Pandey article the melting points, reported from literature given for fungichromin, lagosin, and cogomycin range from 190 to 240° C.
The Merck Index (12th edition) in entry no. 4312 on page 727 mentions the melting point of fungichromin to be 157-162° C. (decomposition).
The subject of the Pandey article is the physicochemical and biological comparison of said three macrolide antibiotics, not the provision of more than 95% pure pentamycin. As stated on page 995 thereof at the beginning of the “Discussion” it was not possible to remove all the minor components even after repeated CCD (countercurrent distribution) purification. The melting points of the thus purified fungichromin, lagosin, and cogomycin are given in Table 2 on page 990 to range from 157-165° C. Nowhere in the Pandey article it is stated that pentamycin has been isolated or crystallized. The only melting points (ranging from 157 to 240° C.!) given in the Pandey article are those stemming from literature sources in Table 1 and those stemming from countercurrent distribution in Table 2. There is, hence, no indication in the Pandey article that more than 95% pure pentamycin has been obtained.
It should be noted that pentamycin is a comparatively large and structurally complicated molecule containing 12 asymmetric centres so that there are 212 (=4096) stereoisomers. In addition, pentamycin contains 5 double bonds which could be subjected to cis-trans-isomerisation. The total chemical synthesis of the right one of the possible 4096 stereoisomers would take years, be an invention on its own, and, from an economic perspective, would be much too costly as compared to the biosynthesis by the Streptomyces strains mentioned above, keeping in mind that the stability of pentamycin is very delicate.
The delicate stability of pentamycin is e.g. evident from R. W. Rickards et al., J. Antibiotics vol. XXIII, no. 12, pages 603-611, describing the aerial autooxidation of lagosin in methanol solution by a radical addition process which can be inhibited by antioxidants. According to Rickards et al. the major primary autooxidation products of lagosin are certain epoxides, while extended autooxidation leads to higher oxidation products and ultimately to polymeric materials. In full accordance with the statements in the above-mentioned Worthen article, Rickards et al. state on page 603 that members of the polyene subgroup of macrolide antibiotics “as a whole are unstable, and exposure to acids, alkalis, heat, air or light is accompanied by decomposition and loss of biological activity. In particular, this sensitivity to air and light, which is primarily associated with the polyene chromophore, creates problems in storage prior to clinical use.”
The above mentioned British patent 884711 states in Example 1 on page 8, line 41, that “pure lagosin” was obtained. Said Example 1 describes the recovery of lagosin from the fermentation broth comprising the steps of extracting the fermentation broth cuttings with butanol, concentrating the extract, adding water, washing with diethyl ether, filtering, extracting with methanol in a soxhlet and filtering whereupon according to page 8, lines 33 and 35, of the British patent lagosin in a purity of “approximately 75%” is obtained. In order to allegedly remove impurities, the 75% pure lagosin is, as described on page 8, lines 34 to 42, extracted in a soxhlet with chloroform, air dried, extracted with ethyl ether, air dried and reextracted with methanol. On cooling the methanol overnight allegedly “pure lagosin” is obtained, but no melting point is provided.
For a person skilled in the art reading the above passage in the British patent it is extremely doubtful that by the proceedings described above the 75% pure lagosin could, in fact be further purified. This is because the treatment with diethyl ether and methanol has already been effected before without increasing the purity of lagosin above 75%. Hence, why should a repetition of said treatment increase purity substantially? In addition, the steps of drying lagosin in the air would be expected to lead to oxidation as described by Rickards et al. (cf. above). Finally, crystallization of crude pentamycin from methanol does not yield more than 95% pure pentamycin (even three times repetitive crystallisations) as evidenced by Example 8 of the present application. This is because, as was found by the present inventors, certain impurities crystallize together with pentamycin and, hence, cannot be removed by crystallization, but only by other means as described in the present invention. It should be kept in mind that pentamycin is a comparatively large molecule so that the impurities may be distinguished from pentamycin just by a tiny little difference, e.g. epoxidation of one of the five double bonds. Certainly, the separation of compounds which are structurally so close to each other poses enormous problems. Although it could already be expected from the above that no “pure” lagosin can be obtained by the procedure of Example 1 of the British patent 884,711, the applicants of the present invention ordered an outside institute to repeat the process described in said Example as closely as possible. The result is described in Example 10 of the present application. As expected, while the purity of “approximately 75%” for the intermediate material could be roughly confirmed (found: 67.3%), the further purification steps did not yield “pure” pentamycin, but only pentamycin in a purity of 70.1%.
Pentamycin was the active ingredient in a drug registered in the 1980s under the trade name Pentacin® in Switzerland, but, due to difficulties in meeting the registered product specifications for purity and stability (even though the marketed formulation contained an antioxidant), was withdrawn from the market. According to the specification in the Swiss registration documents the purity of pentamycin was 95%. However, when using the modern means of analysis available today, it now turned out that a number of impurities present in pentamycin as registered in the past had not been detected and that, hence, the actual purity of pentamycin in the past was much lower than 95%.
Pentamycin itself has a lipophilic and a very hydrophilic part and thus behaves similar to a surfactant. Furthermore it is relatively insoluble in water and tends to form gels, which are almost unfilterable.
Despite intensive efforts, the present inventors were not able to increase the purity of pentamycin above 93% for a long time. In fact, the purity threshold of 93% appeared to be unsurmountable until the present invention.
The problem to be solved by the present inventors was to increase the purity of pentamycin above 93%, especially above 95%, and to bring pentamycin into a form stable enough to enable its reintroduction into the pharmaceutical market in Switzerland and its registration as a drug in other countries.